Previous basic research on Protein C, a naturally occurring plasma protein, has been translated into diagnostic and therapeutic tools now used in the clinic. Activated protein C (APC) exerts two major and distinct activities, (1) anticoagulant activity and (2) initiation of cell signaling that activates multiple cytoprotective activities. The cytoprotective actions include anti-apoptotic and anti-inflammatory activities, favorable alterations of gene expression, and stabilization of endothelial barriers. Whereas the antithrombotic actions of APC have long been appreciated, only very recently has the physiologic and pharmacologic importance of APC's direct effects on cells become apparent. According to a recently established, incomplete paradigm, APC's cell signaling involves binding of APC by endothelial protein C receptor (EPCR) combined with protease activated receptor-1 (PAR1) proteolytic activation. Our preliminary data identify an additional novel cell signaling pathway for APC, namely a reelin-like signaling pathway involving ligation of apolipoprotein E Receptor 2 (apoER2) that initiates signaling via the intracellular adaptor protein, disabled-1 (Dab1), and Src-family kinases (Src and Fyn) with subsequent downstream actions via the phosphatidylinositol-3-kinase (PI3K)-Akt survival pathway. We need to understand mechanisms for APC's cell signaling reactions and its multiple cytoprotective effects. The three Specific Aims of this hypothesis-driven project are: 1) to define the structural basis for binding of APC to apoER2; 2) to prepare and characterize new APC variants with novel mutations that selectively alter APC's targeting of PAR1 and apoER2; and 3) to clarify molecular mechanisms and the roles for each of the various APC receptors responsible each of APC's direct cytoprotective effects on cells. This proposal will address multiple key questions about APC-receptor interactions. The answers from this basic research project may have direct clinical relevance with substantial impact for both basic and clinical research and with obvious clinical implications.